Recording dynamics of cellular processes

ABSTRACT

Devices and methods for recording dynamics of cellular and/or biochemical processes, including a device including one or more dispersive elements configured to receive a pulsed laser beam with a spectrum of different wavelengths and disperse the spectrum of the pulsed laser beam; and one or more first elements configured to receive the dispersed spectrum of the pulsed laser beam, and generate a multiphoton excitation area in a biological sample by re-overlapping in time and space the dispersed spectrum of the pulsed laser beam on an area in the biological sample, wherein the device is configured to record at high speed changes of cellular and biochemical processes of a population of cells of the biological sample based on generation of the multiphoton excitation area in the biological sample.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalPatent Application Ser. No. 62/047,425, filed on Sep. 8, 2014, andentitled “Recording Dynamics of Cellular Processes,” which isincorporated by reference herein in its entirety.

TECHNICAL FIELD

This disclosure relates to recording the dynamics of cellular processes.

BACKGROUND

A biological sample, such as an organism, includes cells that performvarious activities. Other biochemical processes also may be performed bythe organism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate example components of a first system forrecording dynamics of cellular and/or biochemical processes of apopulation of cells of a biological sample.

FIGS. 3-5 illustrate characteristics of an example multiphotonexcitation area applied to a biological sample.

FIGS. 6-8 are flow charts illustrating example processes for recordingat high speed the dynamics of cellular and/or biochemical processes of apopulation of cells of a biological sample.

FIG. 9 illustrates example components of a second system for recordingdynamics of cellular and/or biochemical processes of a population ofcells of a biological sample.

FIG. 10 illustrates example schematics of the multiphoton excitationarea that may be formed in the biological sample.

FIGS. 11-13 are flow charts illustrating example processes for recordingat high speed the dynamics of cellular and/or biochemical processes of apopulation of cells of a biological sample.

Like reference symbols in different figures represent like elements.

DETAILED DESCRIPTION

Recent research in biomedical technologies have been targeted towardsobtaining information about detailed connectivity between cells toperform various activities, for example, how a network of neuronsinterconnect and engage in dynamic activities. It may be useful to comeup with a high-resolution imaging technique that is capable of recordingthe dynamics (also referred to interchangeably as “changes” in thefollowing sections) of cellular or biochemical processes in a biologicalsample, where the technique is capable of recording the dynamics ofsmall or large groups of cells at the resolution of a single cell andwith high-speeds.

Towards this end, this disclosure describes devices, systems andassociated methods for recording the dynamics or changes of cellular orbiochemical processes of a population of cells of a biological sample.The biological sample may correspond to any organism, including, forexample, worms, fish, mammals, or some other suitable organism. Thedynamics of cellular or biochemical processes may include, for example,activity of neurons associated with brain functions, cardiovascularactivities, or some other suitable activity. Sections of the biologicalsample that are to be recorded may be excited using a light source, forexample, a laser beam. The recording may be performed in high-speed, forexample, successive images may be generated or captured simultaneously,or near-simultaneously in an order of seconds or fractions of seconds.The population of cells may include a large population of cells of thebiological sample, for example, in the range of tens to thousands, ormillions, of cells. In some implementations, the population of cells mayencompass an entire organism, and the recording may capture the dynamicsof cellular or biochemical processes of a complete mammal.

To facilitate the recording of changes of the cellular and/orbiochemical processes, molecular reporters may be artificiallyintroduced in the biological sample, for example, in the cells. Therecording of the multiphoton excitation and subsequent optical readoutmay be based on light emission properties of the molecular reporters.

The devices, systems and methods that are described herein for recordingof the dynamics or changes of cellular or biochemical processes of apopulation of cells of a biological sample using molecular reporters mayfind applicability in various situations. For example, the devices,systems and methods may be used to serve applications in neuroscience.Additionally or alternatively, the devices, systems and methods may beconfigured to serve discovery of new drugs, therapeutic agents orstrategies. Additionally or alternatively, the devices system andmethods may be configured to serve applications in stem cell research.Additionally or alternatively, the devices system and methods may beconfigured to serve applications in cancer research.

In a general aspect, a device comprises one or more dispersive elementsthat are configured to receive a pulsed laser beam with a spectrum ofdifferent wavelengths and disperse the spectrum of the pulsed laserbeam. The device also includes one or more first elements that areconfigured to receive the dispersed spectrum of the pulsed laser beam,and generate a multiphoton excitation area in a biological sample byre-overlapping in time and space the dispersed spectrum of the pulsedlaser beam on an area in the biological sample. The device is configuredto record at high speed changes of cellular and biochemical processes ofa population of cells of the biological sample based on generation ofthe multiphoton excitation area in the biological sample.

Particular implementations may include one or more of the followingfeatures. The device may include a first amplifier that is configured toamplify the pulsed laser beam, wherein the pulsed laser beam amplifiedby the first amplifier may be used at the dispersive element. The devicemay include a wavelength tuning element that is configured to tune thecentral wavelength of the pulsed laser beam amplified by the firstamplifier to an excitation wavelength of a molecular reporter associatedwith the biological sample. The device may include a motorized half-waveplate and a polarizing beam splitter with a shutter that are configuredto attenuate the power of the pulsed laser beam at an output of thewavelength tuning element. The first amplifier may include aregenerative amplifier.

The molecular reporter may be configured to facilitate recording of thecellular or biochemical changes of the biological sample. The molecularreporter may include one or more molecules, which also may be referredto as labeling molecules, which may be artificially introduced into thecells. The changes of cellular and biochemical processes of the samplemay be recorded via multiphoton excitation and subsequent opticalreadout of light emission properties of the artificially-introducedmolecules. The molecules may be configured to readout at least one ofcalcium concentrations in the cells, or changes to calciumconcentrations in the cells. The molecules may include proteins that areexpressed by the cells by introducing genetic information to the cells.The molecules may be configured to report on at least one of membranevoltage of the cells, or changes to the membrane voltage of the cells.The molecules may be configured to report on synaptic activity in thecells. The molecules may be configured to report on metabolic activityin the cells. The molecules may be configured to report on enzymaticactivity in the cells. The molecules may be configured to bind to atleast one of naturally available proteins in the cells, or othermolecules in the cells. The molecules may be configured to report on astage of the cell cycle.

The multiphoton excitation area may comprise an arbitrarily-shapedexcitation disc of the dispersed spectrum of the pulsed laser beam.Spectral components of the pulsed laser beam may overlap in time andspace in a focal region of the first element.

A first element may include at least one of a grating, a prism, a lensor a microscope objective lens that are configured to generate themultiphoton excitation area in the biological sample by imaging onto thebiological sample an area on the dispersive element illuminated by thepulsed laser beam.

The high speed recording may include near simultaneous imaging of thecellular or biochemical changes within a single plane of the biologicalsample. The near-simultaneous imaging may include imaging in an order ofseconds to picoseconds.

The multiphoton excitation area may include a wide-field excitation discwith one of a predetermined diameter or a predetermined axialconfinement. The predetermined diameter or the predetermined axialconfinement may be user-configurable. At least one of the predetermineddiameter or the predetermined axial confinement may be in a range thatis in an order of 1 μm to 1 mm.

In another aspect, cellular or biochemical changes of a population ofcells of a biological sample are imaged at high-speed by providing apulsed laser beam with a spectrum of different wavelengths at adispersive element. The spectrum of the pulsed laser beam is dispersedusing the dispersive element. A multiphoton excitation area based on thedispersed spectrum of the pulsed laser beam is generated using a firstelement. The multiphoton excitation area is applied to a biologicalsample using the first element. An imaging detector array is used tocapture information in parallel about the cellular or biochemicalchanges of a population of cells of the biological sample included inthe multiphoton excitation area. The information about the cellular orbiochemical changes is captured at a resolution of a single cell.

Particular implementations may include one or more of the followingfeatures. The pulsed laser beam may be generated using a laser source.The pulsed laser beam may be amplified using a first amplifier. Acentral wavelength of the pulsed laser beam amplified by the firstamplifier may be tuned using a wavelength tuning element to anexcitation wavelength of a molecular reporter that produces a signalassociated with the cellular or biochemical changes of the cells of thebiological sample. The pulsed laser beam tuned by the wavelength tuningelement may be provided at the dispersive element.

The molecular reporter may include molecules, which also may be referredto as labeling molecules, which may be artificially introduced into thecells. The changes of cellular and biochemical processes of the samplemay be recorded via multiphoton excitation and subsequent opticalreadout of light emission properties of the artificially-introducedmolecules.

The molecules may be configured to readout at least one of calciumconcentrations in the cells, or changes to calcium concentrations in thecells. The molecules may include proteins that are expressed by thecells by introducing genetic information to the cells. The molecules maybe configured to report on at least one of membrane voltage of thecells, or changes to the membrane voltage of the cells. The moleculesmay be configured to report on synaptic activity in the cells. Themolecules may be configured to report on metabolic activity in thecells. The molecules may be configured to report on enzymatic activityin the cells. The molecules may be configured to bind to at least one ofnaturally available proteins in the cells, or other molecules in thecells. The molecules may be configured to report on a stage of the cellcycle.

The power of the pulsed laser beam tuned by the wavelength tuningelement may be attenuated using an attenuating element. Across-sectional area of the pulsed laser beam may be modified ormodulated using a second optical element. The pulsed laser beam with themodulated cross-sectional area may be provided at the dispersiveelement.

The attenuating element may include one of a half-wave plate and a beamsplitter, or a shutter. The second optical element may include atelescope.

The first element may include at least one of a relay lens or amicroscope objective that are configured to generate the multiphotonexcitation area in a focal region of the microscope objective.

In another aspect, a device includes one or more manipulating elementsconfigured to manipulate spatial or angular displacement of a pulsedlaser beam that includes a spectrum of different wavelengths. The devicealso includes one or more first elements configured to modify the shapeof the pulsed laser beam. The device further includes one or moredispersive elements configured to disperse the spectrum of the pulsedlaser beam. In addition, the device includes one or more second elementsconfigured to generate a multiphoton excitation area based on thedispersed spectrum of the pulsed laser beam and apply the multiphotonexcitation area to a biological sample. The device is configured torecord at high-speed changes of cellular or biochemical processes of apopulation of cells of the biological sample based on application of themultiphoton excitation area to the biological sample.

Particular implementations may include one or more of the followingfeatures. The device may be configured to perform the high-speedrecording at single-cell spatial resolution for a predetermined timeperiod.

A first element may include a spherical lens that is configured to shapean area on the dispersive element used by the manipulating device togenerate a trajectory on the dispersive element that corresponds to themultiphoton excitation area in the biological sample. Recording of thecellular or biochemical changes of cells of the biological sample may beperformed in two dimensions based on sequential or parallel excitationthat may be limited at a time to the area in the biological samplecorresponding to the generated trajectory. The generated trajectory mayinclude a spiral path.

The multiphoton excitation area may include an arbitrarily-shapedwide-field excitation area with a predetermined axial confinement thatis user-configurable in a range in an order of 1 μm to 1 mm. Thearbitrarily-shaped excitation area may include a predetermined diameterthat may be user-configurable. The predetermined diameter may be in arange that is in an order of 1 μm to 1 mm. The arbitrarily-shapedexcitation area may include a variable diameter.

The device may comprise a detection section for detecting the changes ofthe cellular or biochemical processes of the cells of the biologicalsample. The detection section may include one or more third elementsconfigured to separate a signal associated with the changes of thecellular or biochemical processes from the pulsed laser beam. A thirdelement may include a dichroic mirror.

The detection section may include an array detector that is configuredto generate images of the cellular or biochemical changes of thepopulation of cells of the biological sample.

Implementations of the above techniques include methods, systems,computer program products and computer-readable media. One such systemcomprises one or more devices and/or components that are configured torecord at high speed the changes of cellular and/or biochemicalprocesses of cells of a biological sample based on generation of amultiphoton excitation area in the biological sample. The system alsocomprises a machine-readable medium for storing instructions that areexecutable by a processing unit and, when executed by the processingunit, are configured to cause the devices and/or components to performone or more of the above described operations.

One such method involves performing one or more of the above describedoperations using devices and components for recording the changes ofcellular and/or biochemical processes of a population of cells of abiological sample.

One such computer program product is suitably embodied in anon-transitory machine-readable medium that stores instructionsexecutable by one or more processing units. The instructions areconfigured to cause the one or more processing units to perform one ormore of the above described operations. One such computer-readablemedium stores instructions that, when executed by a processing unit, areconfigured to cause the processing unit to perform one or more of theabove described operations.

The details of one or more disclosed implementations are set forth inthe accompanying drawings and the description below. Other features,aspects, and advantages will become apparent from the description, thedrawings and the claims.

FIGS. 1 and 2 illustrate example components of a first system 100 forrecording dynamics of cellular and/or biochemical processes of apopulation of cells of a biological sample. As shown in FIG. 1, thesystem 100 includes a device that is configured to record the dynamicsof cellular and/or biochemical processes of the population of cells ofthe biological sample at high speed based on generation of a multiphotonexcitation area in the biological sample. The device includes adispersive element 102 and an optical element 104. The system 100 alsoincludes additional components that are used for generation of themultiphoton excitation area in the biological sample, such as a lasersource 106, an amplifier 108, a tuning element 110, and an opticalelement 112. In addition, the device of system 100 includes componentsthat are used for recording the dynamics of the cellular and biochemicalprocesses, such as an optical separator 122, a splitter 124, a detector126 and an intensifier 128. Furthermore, the system 100 includes atarget device 140 and a molecular reporter that is applied to thebiological sample.

The dispersive element 102 is configured to disperse the spectrum of alight source. For example, the dispersive element may disperse thespectrum of a pulsed laser beam that is applied to the biologicalsample. The pulsed laser beam may include a spectrum of differentwavelengths. The undispersed spectrum of the pulsed laser beam asreceived at the dispersive element is indicated by 152, while thedispersed spectrum of a pulsed laser beam following processing by thedispersive element is indicated by 154. The pulses 152, 154, 156, 158and 160 indicate geometric dispersion of the pulsed laser beam in intemporal focusing as a function of axial position. In someimplementations, there may be several dispersive elements 102 includedin the device of system 100.

In some implementations, the dispersive element may spatially dispersethe spectrum of the pulsed laser beam. In some implementations, thepulsed laser beam that is dispersed by the dispersive element may have apicosecond pulse or a femtosecond pulse, or some other suitable pulseduration. The dispersive element may be a grating, a prism, or someother suitable component.

The optical element 104 is configured to refocus the dispersed spectrumof the pulsed laser beam in an area of the biological sample, therebygenerating a multiphoton excitation area in the biological sample. Thedispersed spectrum of the pulsed laser beam processed by the opticalelement is indicated by 156. In some implementations, there may beseveral optical elements 104 included in the device of system 100.

The optical element 104 may comprise one or more discrete opticalelements. In some implementations, the optical element 104 includes alens (for example, a relay lens) and a microscope objective. In someother implementations, the optical element 104 includes a grating, aprism, or some other suitable element. The optical element 104 images anilluminated spot on the dispersive element in the biological sample. Indoing so, the spectral components of the pulsed laser beam areconfigured to overlap in time and/or space in the focal region of theoptical element 104, leading to the multiphoton excitation. Outside thefocal region of the optical element, the spectral components of thepulsed laser beam do not overlap in time and/or space, thereby reducingthe probability of multiphoton excitation. In some implementations, themultiphoton excitation area that is generated in the biological sampleincludes a two-photon excitation area. In some implementations, theexcitation area includes an excitation disc. For example, a wide-fieldtwo-photon excitation area of approximately 60 μm in diameter with anaxial confinement of approximately 1.9 μm may be generated in oneimplementation.

In some implementations, the laser source 106 is configured to generatethe pulsed laser beam that is used for the generation of the multiphotonexcitation area. In some implementations, the laser source 106 mayinclude a titanium-sapphire (Ti:Sa) laser source that is configured togenerate a laser beam with a pulse duration in the order of picosecondsor femtoseconds.

The amplifier 108 is configured to amplify the pulsed laser beamgenerated by the laser source 106. In doing so, the amplifier 108 mayincrease the power of the pulsed laser beam, for example, generatinghigher peak pulse energy that may be useful for generating themultiphoton excitation area. For example, the power of the pulsed laserbeam at the output of the amplifier 108 may be in an order ofmilli-Joules. In some implementations, the amplifier 108 includes aregenerative amplifier. The regenerative amplifier may have alow-repetition rate.

In some implementations, the tuning element 110 is a wavelength tuningelement that is configured to tune the pulsed laser beam amplified bythe amplifier 108 to an excitation wavelength of a molecular reporterassociated with the biological sample. In some implementations, thetuning element 110 is configured to tune the central wavelength of thepulsed laser beam. In some implementations, the tuning element 110includes an optical parametric amplifier (OPA). In some implementations,the tuning element 110 also includes a motorized half-wave plate and apolarizing beam splitter with a shutter that are configured to attenuatethe power of the pulsed laser beam at an output of the tuning element.For example, the laser power at the output of the OPA may be in an orderof 20 micro-Joules (μJ), which is attenuated by the motorized half-waveplate and a polarizing beam splitter to an order of 2 μJ.

The molecular reporter may include artificially-introduced molecules,also referred to as labeling molecules in some implementations, whichare configured to facilitate recording of the cellular or biochemicaldynamics of the biological sample. The recording of the multiphotonexcitation and subsequent optical readout may be based on light emissionproperties of the labeling molecules. In some implementations, themolecular reporter or labeling molecules may be included as part of thedevice that comprises the dispersive element 102 and the optical element104.

In some implementations, the labeling molecules are configured toreadout calcium concentrations in the cells, and/or changes to calciumconcentrations in the cells. For example, the molecular reporter may bea nuclear-localized, genetically encoded calcium indicator such as, butnot limited to, NLS-GCaMP5K, which enables recording the dynamics ofindividual cells of the biological sample.

In some implementations, the labeling molecules include proteins thatare expressed by the cells by introducing genetic information to thecells. In some implementations, the labeling molecules are configured tobind to naturally available proteins, and/or other molecules, in thecells.

In some implementations, the labeling molecules are configured to reporton membrane voltage of the cells, and/or changes to membrane voltage ofthe cells. In some implementations, the labeling molecules areconfigured to report on synaptic activity in the cells. In someimplementations, the labeling molecules are configured to report onmetabolic activity in the cells. In some implementations, the labelingmolecules are configured to report on enzymatic activity in the cells.In some implementations, the labeling molecules are configured to reporton a stage of the cell cycle.

The optical element 112 is configured to adjust the cross-sectional areaof the pulsed laser beam at an output of the tuning element 110. Forexample, the optical element 112 may include a telescope comprising oneor more lenses 112 a and 112 b. In some implementations, the telescopemay expand the diameter of the pulsed laser beam from diameter 172 a todiameter 172 b, and further to diameter 172 c that is applied on to thedispersive element 102. In some other implementations, the opticalelement 112 may contract the diameter of the pulsed laser beam, oradjust the shape of the pulsed laser beam in some other suitable form.Based on this adjustment, the multiphoton excitation area may beconfigured to have an arbitrary shape. In some implementations, theremay be several optical elements 112 included in the system 100.

In some implementations, one or more of the laser source 106, theamplifier 108, the tuning element 110, and the optical element 112 areincluded as parts of the device configured to record the dynamics ofcellular and/or biochemical processes of the population of cells of thebiological sample, in addition to the dispersive element 102 and theoptical element 104. In some implementations, these components form animaging section of the device.

The optical separator 122 is configured to separate a signal associatedwith the cellular or biochemical dynamics of the biological sample fromthe pulsed laser beam. For example, the optical separator 122 separatesa fluorescence signal corresponding to the molecular reporter from theexcitation light associated with the multiphoton excitation area. Theexcitation light is generated by the pulsed laser beam. In someimplementations, the optical separator includes a dichroic mirror and amultiphoton filter 122 b.

The splitter 124 is configured to split into different channels thesignal that is associated with the cellular or biochemical processes andseparated by the optical separator 122. In some implementations, thesplitter 124 includes a wavelength separator composed of a dichroicmirror 124 a and a bandpass filter 124 b that splits the fluorescencesignal into two channels.

The detector 126 is configured to generate images of the cellular orbiochemical dynamics of the biological sample. In some implementations,the detector includes an image capture device, such as a scientificcomplementary metal-oxide semiconductor (sCMOS) camera or an electronmultiplying charge coupled device (EMCCD) camera, or some other suitableparallel image capture device. In some implementations, the imagesgenerated by the detector 126 are captured and/or processed usingcustom-written programming scripts, for example, scripts written using asuitable programming language such as LabVIEW™, or Andor Solis Basic™,or some other suitable script. The images captured and/or processedusing these scripts may be stored in computer memory for subsequentprocessing, for example, using a processing device such as a computer.

The intensifier 128 is configured to record the cellular or biochemicalsignal associated with the multiphoton excitation area and relay theoutput of the intensifier onto the detector 126. In someimplementations, the intensifier 128 includes a high-gain imageintensifier that records the fluorescence signal in a wide-field manner.The intensifier 128 further includes a relay lens to image the recordedfluorescence signal on to the detector. This enables the intensifier tomaintain a high signal-to-noise ratio even at short exposure times (forexample, exposure times on the order of 10 milliseconds).

In some implementations, one of more of the optical separator 122, thesplitter 124, the detector 126 and the intensifier 128 are included asparts of a detection section of the device configured to record thedynamics of cellular and/or biochemical processes of the population ofcells of the biological sample, in addition to the components of theimaging section of the device. In some implementations, the device andother components of the system 100 may be interfaced with existingmicroscopes and/or light sources for recording the dynamics of cellularor biochemical processes.

The target device 140 includes the biological sample. In someimplementations, the dimensions of the target device may be differentfrom that shown in FIG. 1. For example, the target device may be largeenough to accommodate an entire organism, such as a mammal. FIG. 2 showsdetails of an example target device 140. As shown, the target device maybe a microfluidic chip. The target device 140 includes a holding section142 where the biological sample is placed. The multiphoton excitationarea applied to the target device is indicated by 144.

In some implementations, a fluid (for example, but not limited to,oxygen) is supplied to the target device to excite the biologicalsample. The fluid may be supplied in the chamber 146.

In some implementations, the device and other components of system 100may be used to serve applications in neuroscience, for example, but notlimited to, mapping chemosensory neuronal circuits in a nervous system.The system 100 may combine controlled sensory stimulation with unbiasedfast volumetric neuronal recording capable of capturing, at single-cellresolution, the activity of the majority of the neurons in the brain ofan entire organism. Additionally or alternatively, the device andcomponents of system 100 may be configured to serve discovery of one ormore drugs, therapeutic agents, or therapeutic strategies. Additionallyor alternatively, the device and components of system 100 may beconfigured to serve applications in stem cell research. Additionally oralternatively, the device and components of system 100 may be configuredto serve applications in cancer research.

FIGS. 3-5 illustrate characteristics of an example multiphotonexcitation area applied to a biological sample. The multiphotonexcitation area shown in these figures may be generated using the system100. FIG. 3 shows a two-dimensional cross-section 200A of a portion ofthe biological sample that is treated using the system 100. In theillustrated example, the cross-section encompasses 512×512 squarepixels. The multiphoton excitation area 202 is formed in the biologicalsample encompassing a population of cells (such as, but not limited to,neurons). The Gaussian intensity pixel distribution of the fluorescencesignal associated with the multiphoton excitation area 202 in the x-axisis indicated by 206, and in the y-axis is indicated by 204.

FIG. 5 illustrates the intensity of the fluorescence signal 222associated with the molecular reporter that is generated due to thedynamics of the cellular and/or biochemical processes of the biologicalsample. The fluorescence signal 222 may be recorded using the system100. The cross-sectional area 200C may be similar to the cross-section200A of the portion of the biological sample that is treated using thesystem 100. The multiphoton excitation area 202 may be applied to thebiological sample to record the fluorescence signal 222.

The system 100 may be applied to perform three-dimensional imaging ofneuronal activity in a large portion of the brain of an organism. Anexample of such a system and results obtained by the example system areillustrated in FIGS. X1, X2, X3, X4 and X5 of U.S. Provisional PatentApplication Ser. No. 62/047,425, along with their associateddescription, which was incorporated by reference above. In U.S.Provisional Patent Application Ser. No. 62/047,425, FIGS. X1(a)-X1(f)illustrate volumetric fluorescence imaging using wide-field two-photonlight sculpting, FIGS. X2(a)-X2(r) illustrate in vivo characterizationof NLS-GCaMP5K, FIGS. X3(a)-X3(d) illustrate brain-wide wide-fieldtemporal focusing (WF-TeFo) Ca²⁺ imaging in C. elegans, FIGS.X4(a)-X4(e) illustrate time-series correlations between neurons, andFIGS. X5(a)-X5(f) illustrate WF-TeFo Ca²⁺ imaging in worms duringchemosensory stimulation. The system 100 may be similarly applied toperform high-speed (such as simultaneous and/or near-simultaneous) two-or three-dimensional imaging of cellular or biochemical activity of anyother organism. The images may be generated and/or recorded for apopulation of cells ranging from tens to millions, with the imagingperformed at a resolution of a single-cell.

FIGS. 6-8 are flow charts illustrating example processes 300A, 300B and300C for recording at high speed the dynamics of cellular and/orbiochemical processes of a population of cells of a biological sample.The processes 300A, 300B or 300C, or some suitable combination of these,may be used to image the cellular or biochemical activity of an organismusing the system 100. Accordingly, the following section describes theprocesses 300A, 300B and 300C as being performed by components of thesystem 100. However, the processes also may be performed by othersystems or system configurations.

In some implementations, one or more of the processes 300A, 300B and/or300C are implemented using a processing device, for example, a computer,a server, a mechanized device such as a robot, or some other suitabledevice. The processing device may include one or more processing unitsthat execute instructions for operating the various components of thesystem 100 to generate the multiphoton excitation area in the biologicalsample, and record images of the dynamics of cellular and/or biochemicalprocesses of cells of the biological sample. The processing unit may bea microprocessor or microcontroller, a field-programmable gate array(FPGA), a digital signal processor (DSP), or some other suitable unitthat is capable of processing instructions and/or data.

In some implementations, the instructions and recorded data (such as thecaptured images) may be stored in memory associated with the processingdevice. The memory may be one of a hard disk, flash memory, read-onlymemory (ROM), random access memory (RAM), or some suitable combinationof these, or some other memory that is capable of storing instructionsand/or data.

In some implementations, the instructions may be configured by a user,such as, but not limited to, an operator of the system 100 using a userinput interface (for example, a keyboard and/or a mouse coupled to theprocessing device). The instructions may be configured and/or generatedusing some suitable form, for example, a programming language or scriptsuch as LabVIEW™, Andor Solis Basic™, MetaMorph™, Molecular Devices™,Universal Imaging™, or some other suitable method.

The process 300A illustrated in FIG. 6 describes recording at high speedthe dynamics of cellular and/or biochemical processes of a population ofcells of a biological sample. At 302, a pulsed laser beam is provided ona dispersive element. For example, a pulsed laser beam may be providedat an area of the dispersive element 102. The pulsed laser beam may begenerated by the laser source 106, amplified by the amplifier 108, andtuned by the tuning element 110. The shape of the pulsed laser beam alsomay be adjusted by the optical element 112 before being provided on thedispersive element 102.

The spectrum of the pulsed laser beam is dispersed at 304. For example,the dispersive element 102 may spatially disperse the spectrum of thepulsed laser beam. By dispersing the spectrum of the pulsed laser beam,the dispersive element 102 may split the pulsed laser beam into itsindividual component wavelengths.

At 306, a multiphoton excitation area is generated using the pulsedlaser beam. For example, the optical element 104 refocuses the pulsedlaser beam dispersed by the dispersive element 102 into the focal regionof the optical element 104, generating the multiphoton excitation area.

At 308, the multiphoton excitation area is applied to a biologicalsample. For example, the optical element 104 images the multiphotonexcitation area in a biological sample that is placed in the targetdevice 140. The multiphoton excitation area imaged in the biologicalsample may be similar to the multiphoton excitation area 202.

Information about the cellular or biochemical dynamics of a cellpopulation of the biological sample is captured at single-cellresolution at 310. For example, one or more of the components 122, 124,126, or 128 may be used to record information about cellular or otherbiochemical activity of a population of cells of the biological samplein the target device 140. The information, such as images, may berecorded for the multiphoton excitation area that is imaged in thebiological sample using one or more of the components 102, 104, 106,108, 110, or 112.

The process 300B illustrated in FIG. 7 describes the generation andprocessing of the pulsed laser beam before dispersion by the dispersiveelement. In some implementations, the process 300B may be used inconjunction with the process 300A, while in other implementations theymay be used separately.

At 322, the pulsed laser beam is generated using a laser source. Forexample, the laser source 106 may be operated to generate a laser beamwith a pulse duration in the range of picoseconds or femtoseconds.

The pulsed laser beam is amplified at 324. For example, the amplifier108 may be used to receive the pulsed laser beam generated by the lasersource 106 and increase the power of the pulsed laser beam.

The pulsed laser beam may be tuned to an excitation wavelength of amolecular reporter associated with the biological sample at 326. Forexample, the tuning element 110 may tune the pulsed laser beam amplifiedby the amplifier 108 to an excitation wavelength of the molecularreporter (for example, but not limited to, NLS-GCaMP5K) that is used tonote the changes associated with the cells or biochemical processes ofthe biological sample.

In some implementations, the power of the pulsed laser beam isattenuated at 328. For example, a motorized half-wave plate and apolarizing beam splitter with a shutter that are coupled to the tuningelement 110 may be used to attenuate the power of the pulsed laser beamat an output of the tuning element to a range in the order of units ofmicro-Joules.

In some implementations, the cross-sectional area of the pulsed laserbeam is expanded at 330. For example, the optical element 112 may beused to adjust the cross-sectional area of the pulsed laser beam at anoutput of the tuning element 110. The optical element 112 may expand thediameter of the pulsed laser beam from diameter in some implementations,while in other implementations, the optical element may contract thediameter of the pulsed laser beam, or adjust the shape of the pulsedlaser beam in some other arbitrary form.

The pulsed laser beam is provided for dispersion at 332. For example,the optical element 112 may illuminate an area of the dispersive element102 using the pulsed laser beam with the adjusted cross-sectional area.

The process 300C illustrated in FIG. 8 describes the detection andrecording of information about the dynamics of cellular or biochemicalprocesses of the biological sample based on the multiphoton excitationarea formed in the biological sample. In some implementations, theprocess 300C may be used in conjunction with the process 300A or theprocess 300B, or both, while in other implementations the differentprocesses may be used separately.

At 342, the signal associated with cellular dynamics of the biologicalsample is separated from the excitation light. For example, the opticalseparator 122 may separate a fluorescence signal corresponding to themolecular reporter used with the biological sample from the excitationlight generated by the pulsed laser beam. As described previously, themolecular reporter may include one or more molecules, such as labelingmolecules, which are artificially introduced into the cells. The changesof cellular and biochemical processes of the sample may be recorded viamultiphoton excitation and subsequent optical readout of light emissionproperties of the artificially-introduced molecules.

The molecules may be configured to readout at least one of calciumconcentrations in the cells, or changes to calcium concentrations in thecells. The molecules may include proteins that are expressed by thecells by introducing genetic information to the cells. The molecules maybe configured to report on at least one of membrane voltage of thecells, or changes to the membrane voltage of the cells. The moleculesmay be configured to report on synaptic activity in the cells. Themolecules may be configured to report on metabolic activity in thecells. The molecules may be configured to report on enzymatic activityin the cells. The molecules may be configured to bind to at least one ofnaturally available proteins in the cells, or other molecules in thecells. The molecules may be configured to report on a stage of the cellcycle.

The separated signal is split into different channels at 344. Forexample, the splitter 124 may split the fluorescence signal separated bythe optical separator 122 into different channels.

The signal is recorded at 346. For example, the intensifier 128 mayrecord, corresponding to the multiphoton excitation area, thefluorescence signal associated with the molecular reporter.

At 348, information about the cellular or biochemical processes of thebiological sample is captured at single-cell resolution. For example,the intensifier may relay the recorded fluorescence signal onto thedetector 126. The detector 126 may generate images of the dynamics ofcellular or biochemical processes of the biological sample using animage sensor. The images may be generated with a resolution in the orderof a single cell. These images may be captured using scripts andprocessed, or stored for post-processing.

FIG. 9 illustrates example components of a second system 400 forrecording changes of cellular and/or biochemical processes of apopulation of cells of a biological sample. The system 400 includes adevice that is configured to record the dynamics of cellular and/orbiochemical processes of the population of cells of the biologicalsample at high speed (such as simultaneous or near-simultaneous in theorder of nanoseconds or picoseconds) based on generation of amultiphoton excitation area in the biological sample. The deviceincludes a dispersive element 406 and a first element 408. The firstelement 408 includes one or more components, such as lens 408 a and/ormicroscope objective 408 b.

The system 400 also includes additional components that are used forgeneration of the multiphoton excitation area in the biological sample,such as laser sources 410 and/or 412. In addition, the device of system400 includes components that are used for recording the dynamics of thecellular and biochemical processes, such as a second element 422, athird element 424, a detector 426 and an intensifier 428. Furthermore,the system 100 includes a target device 432. In some implementations,the system includes a molecular reporter that is used for facilitatingthe recording of the changes of cellular and/or biochemical processes ofthe biological sample.

The dispersive element 406 is configured to disperse the spectrum of alight source. For example, the dispersive element may disperse thespectrum of the pulsed laser beam that is applied to the biologicalsample. The undispersed spectrum of the pulsed laser beam as received atthe dispersive element is indicated by 452, while the dispersed spectrumof a pulsed laser beam following processing by the dispersive element isindicated by 454.

In some implementations, the dispersive element 406 may spatiallydisperse the spectrum of the pulsed laser beam. In some implementations,the pulsed laser beam that is dispersed by the dispersive element mayhave a picosecond pulse or a femtosecond pulse, or some other suitablepulse duration. The dispersive element 406 may be a grating, a prism, orsome other suitable component. In some implementations, there may beseveral dispersive elements 406 included in the device of system 400.

The first element 408 is configured to refocus the dispersed spectrum ofthe pulsed laser beam in an area of the biological sample, therebygenerating a multiphoton excitation area in the biological sample. Insome implementations, the first element 408 includes a lens 408 a (forexample, a relay lens) and a microscope objective 408 b. The lens 408 ain conjunction with the microscope objective 408 b images an illuminatedspot on the dispersive element 406 in the biological sample. In doingso, the spectral components of the pulsed laser beam are configured tooverlap in time and/or space in the focal region of the optical element104, leading to the multiphoton excitation. Outside the focal region ofthe optical element, the spectral components of the pulsed laser beam donot overlap in time and/or space, thereby reducing the probability ofmultiphoton excitation. In some implementations, there may be severalfirst elements 408 included in the device of system 400.

In some implementations, the multiphoton excitation area includes anarbitrarily-shaped excitation area with a predetermined axialconfinement. In some implementations, the arbitrarily-shaped excitationarea may include a predetermined diameter, while in otherimplementations, the arbitrarily-shaped excitation area may include avariable diameter.

In some implementations, the system 400 includes one or more lasersources 410 and/or 412, which are configured to generate the pulsedlaser beam for the multiphoton excitation area. In some implementations,the laser source 410 and/or the laser source 412 may include atitanium-sapphire (Ti:Sa) laser source that is configured to generate alaser beam with a pulse duration in the order of picoseconds or femtoseconds. In some implementations, the laser source 410 and/or the lasersource 412 may include a regenerative amplifier in combination with anoptical system that is configured to tune the wavelength of the pulsedlaser beam. In some implementations, the system 400 may switch betweenthe laser source 410 and the laser source 412 using a flip mirror 414.

In some implementations, one or more of the laser source 410 and/or thelaser source 412 are included as parts of the device configured torecord the dynamics of cellular and/or biochemical processes of thepopulation of cells of the biological sample, in addition to thedispersive element 406 and the first element 408. In someimplementations, these components form an imaging section of the device.

The second element 422 is configured to separate a signal associatedwith the cellular or biochemical dynamics of the biological sample fromthe pulsed laser beam. For example, the optical separator 422 separatesa fluorescence signal corresponding to the molecular reporter associatedwith the cellular dynamics from the excitation light associated with themultiphoton excitation area. The excitation light is generated by thepulsed laser beam. In some implementations, the optical separatorincludes a dichroic mirror.

The third element 424 is configured to propagate the signal separated bythe second element 422 onto a detection plane of a detector 426. In someimplementations, the fourth element 426 includes a tube lens.

The detector 426 is configured to generate images of the cellular orbiochemical dynamics of the biological sample. In some implementations,the detector 426 includes an image capture device, such as a scientificcomplementary metal-oxide semiconductor (sCMOS) camera or an electronmultiplying charge coupled device (EMCCD) camera, or some other suitableimage capture device. In some implementations, the images generated bythe detector 426 are captured and/or processed using custom-writtenprogramming scripts, for example, scripts written using a suitableprogramming language such as LabVIEW™, or Andor Solis Basic™, or someother suitable script. The images captured and/or processed using thesescripts may be stored in computer memory for subsequent processing, forexample, but not limited to, using a processing device such as acomputer.

The intensifier 428 is configured to record the cellular or biochemicalsignal associated with the multiphoton excitation area and relay theoutput of the intensifier onto the detector 426. In someimplementations, the intensifier 428 includes a high-gain imageintensifier for recording the fluorescence signal.

In some implementations, the cellular or biochemical signal associatedwith the multiphoton excitation area is generated by a molecularreporter that is artificially introduced into the cells. The molecularreporter may include molecules, such as labeling molecules, which may beconfigured to facilitate recording of the cellular or biochemicalchanges of the biological sample. The recording of the multiphotonexcitation and subsequent optical readout may be based on light emissionproperties (such as the fluorescence signal) of theartificially-introduced molecules. In some implementations, theartificially-introduced molecules may be included as part of the devicethat includes the dispersive element 406 and the first element 408.

In some implementations, the artificially-introduced molecules areconfigured to readout calcium concentrations in the cells, and/orchanges to calcium concentrations in the cells. For example, themolecular reporter may be a nuclear-localized, genetically encodedcalcium indicator such as, but not limited to, NLS-GCaMP5K, whichenables recording the dynamics of individual cells of the biologicalsample.

In some implementations, the molecules include proteins that areexpressed by the cells by introducing genetic information to the cells.In some implementations, the molecules are configured to bind tonaturally available proteins, and/or other molecules, in the cells.

In some implementations, the molecules are configured to report onmembrane voltage of the cells, and/or changes to membrane voltage of thecells. In some implementations, the molecules are configured to reporton synaptic activity in the cells. In some implementations, themolecules are configured to report on metabolic activity in the cells.In some implementations, the molecules are configured to report onenzymatic activity in the cells. In some implementations, the moleculesare configured to report on a stage of the cell cycle.

In some implementations, the detector 426 includes an imaging sensorwhere the images of the cellular or biochemical process activity aregenerated. The imaging sensor may be a multi-pixel sensor array.

In some implementations, the detector 426 also includes a sequentialreadout element, such as a rolling shutter, that is configured to exposea slit portion of the imaging sensor at a time, wherein the slit portionincludes a predetermined number of pixel rows and is configured to moveover the imaging sensor. This may be the case, for example, when thefirst element used is a cylindrical lens that forms a line ofmultiphoton excitation in the biological sample.

In some implementations, one of more of the second element 422, thethird element 424, the detector 426 and the intensifier 428 are includedas parts of a detection section of the device that is configured torecord the dynamics of cellular and/or biochemical processes of thepopulation of cells of the biological sample, in addition to thecomponents of the imaging section of the device described above. In someimplementations, the device and other components of the system 400 maybe interfaced with existing microscopes and/or light sources forrecording the dynamics of cellular or biochemical processes.

The target device 432 includes the biological sample. In someimplementations, the target device 432 may be large enough toaccommodate an entire organism, such as a mammal.

As described above, the device comprising the dispersive element 406 andthe first element 408, along with other components of the system 400,may be used to record the dynamics of cellular or biochemical processesof the biological sample at a high speed (such as simultaneously ornear-simultaneously in the order of nanoseconds or picoseconds). Thedevice may be configured to perform the high-speed recording atsingle-cell spatial resolution for a predetermined time period. Thepopulation of cells that are recorded may be in an order of tens tothousands, or millions, of cells.

In some implementations, the device and components of system 400described above may be used to serve applications in neuroscience, forexample, but not limited to, mapping chemosensory neuronal circuits in anervous system. The system 400 may combine controlled sensorystimulation with unbiased fast volumetric neuronal recording capable ofcapturing, at single-cell resolution, the activity of the majority ofthe neurons in the brain of an entire organism. Additionally oralternatively, the device and components of system 400 may be configuredto serve discovery of one or more drugs, therapeutic agents orstrategies. Additionally or alternatively, the device and components ofsystem 400 may be configured to serve applications in stem cellresearch. Additionally or alternatively, the device and components ofsystem 400 may be configured to serve applications in cancer research.

FIG. 10 illustrates example schematics of the multiphoton excitationarea that may be formed in the biological sample. The multiphotonexcitation area shown by the schematics may be formed by the system 400.As shown, the schematic 502 a corresponds to a wide-field multiphotonexcitation area. 502 b illustrates the wide-field excitation patternformed in the biological sample in this configuration.

An example of the use of the system 400 for imaging a multiphotonexcitation area in a biological sample and recording the dynamics of thecellular and/or biochemical processes of the biological sample, isillustrated in FIGS. 7, 8, 9, 10 and 11 of U.S. Provisional PatentApplication Ser. No. 62/047,425, along with their associateddescription, which was incorporated by reference above. In U.S.Provisional Patent Application Ser. No. 62/047,425, FIGS. 7A-7Dillustrate an experimental setup and various modalities of lightsculpting microscopy, FIGS. 8A-8D illustrate trading off area versusimaging speed and fluorescence for different excitation modalities,FIGS. 9A-9C illustrate Ca²⁺-imaging f acute mouse brain slices atvarious imaging depths and using different TeFo excitation modalities,FIGS. 10A-10G illustrate theoretical estimation of the rolling shuttereffect on image quality in scattering media, and FIGS. 11A-11Iillustrate experimental demonstration of improved rejection ofscattering by using rolling shutter Ca²⁺-imaging. As illustrated inthese figures and the accompanying description, the system 400 may beapplied to perform high-speed (such as simultaneous and/ornear-simultaneous) two- or three-dimensional imaging of cellular orbiochemical activity of an organism, where the multiphoton excitationarea imaged in the organism may be of an arbitrary shape. The images maybe generated and/or recorded for a population of cells ranging from tensto millions, with the imaging performed at a resolution of asingle-cell.

FIGS. 11-13 are flow charts illustrating example processes 600A, 600B,and 600C for recording at high speed the dynamics of cellular and/orbiochemical processes of a population of cells of a biological sample.The processes 600A, 600B, or 600C, or some suitable combination ofthese, may be used to image the cellular or biochemical activity of anarbitrarily-shaped excitation area of an organism using the system 400.Accordingly, the following section describes the processes 600A, 600B,or 600C as being performed by components of the system 400. However, theprocesses also may be performed by other systems or systemconfigurations.

In some implementations, one or more of the processes 600A, 600B, and/or600C are implemented using a processing device, for example, but notlimited to, a computer, a server, a mechanized device such as a robot,or some other suitable device. The processing device may include one ormore processing units that execute instructions for operating thevarious components of the system 400 to generate the multiphotonexcitation area in the biological sample, and record images of thedynamics of cellular and/or biochemical processes of cells of thebiological sample. The processing unit may be a microprocessor ormicrocontroller, a field-programmable gate array (FPGA), a digitalsignal processor (DSP), or some other suitable unit that is capable ofprocessing instructions and/or data.

In some implementations, the instructions and recorded data (such as thecaptured images) may be stored in memory associated with the processingdevice. The memory may be one of a hard disk, flash memory, read-onlymemory (ROM), random access memory (RAM), or some suitable combinationof these, or some other memory that is capable of storing instructionsand/or data.

In some implementations, the instructions may be configured by a user,for example, but not limited to, an operator of the system 400 using auser input interface (for example, a keyboard and/or a mouse coupled tothe processing device). The instructions may be configured using somesuitable form, for example, a programming language or script such asLabVIEW™, Andor Solis Basic™, MetaMorph™, Molecular Devices™, UniversalImaging™, or some other suitable method.

The process 600A illustrated in FIG. 11 describes recording at highspeed the dynamics of cellular and/or biochemical processes of apopulation of cells of a biological sample. At 602, the displacement ofa pulsed laser beam is manipulated. The pulsed laser beam may begenerated by the laser source 410 and/or 412.

The shape of the pulsed laser beam may be modified at 604. There may besome lens to obtain an arbitrary shape.

At 606, the spectrum of the pulsed laser beam is dispersed. For example,the dispersive element 406 may spatially disperse the spectrum of thepulsed laser beam that is applied to the biological sample.

A multiphoton excitation area is generated using the pulsed laser beamat 608. For example, the first element 408 refocuses the spectrum of thepulsed laser beam dispersed by the dispersive element 406 into the focalregion of the first element 408, generating the multiphoton excitationarea.

The multiphoton excitation area is applied to a biological sample at610. For example, the first element 408 images the multiphotonexcitation area in a biological sample that is placed in the targetdevice 432. The multiphoton excitation area imaged in the biologicalsample may correspond to, for example, one of the multiphoton excitationpatterns 502 b, 504 b or 506 b, depending on the configuration of thefirst element 404.

At 612, information about the cellular or biochemical dynamics of thebiological sample is captured at single-cell resolution in synchronywith the shape and spatial displacement of the pulsed laser beam. Forexample, one or more of the components 422, 424, 426 or 428 may be usedto record information about cellular or other biochemical activity of apopulation of cells of the biological sample in the target device 432.The information, for example, images, may be recorded for themultiphoton excitation area that is imaged in the biological sampleusing one or more of the components 406, 408, 410 or 412.

The process 600B illustrated in FIG. 12 describes the generation andprocessing of the pulsed laser beam before manipulation by themanipulating element. In some implementations, the process 600B may beused in conjunction with the process 600A, while in otherimplementations they may be used separately.

At 622, the pulsed laser beam is generated using a laser source. Forexample, the laser source 410 may be operated to generate a laser beamwith a pulse duration in the range of picoseconds or femtoseconds.

The pulsed laser beam is amplified at 624. For example, the pulsed laseramplifier 412 may include a regenerative amplifier that may be used toreceive the pulsed laser beam generated by the laser source 410 andincrease the power of the pulsed laser beam.

In some implementations, the pulsed laser beam may be tuned to anexcitation wavelength of a molecular reporter associated with thebiological sample at 626. For example, the system 400 may includeoptical elements 416 a and/or 416 b that may be used to tune the pulsedlaser beam to an excitation wavelength of the molecular reporter (forexample, but not limited to, NLS-GCaMP5K) that is used to note theactivity associated with the cells or biochemical processes of thebiological sample.

In some implementations, the power of the pulsed laser beam isattenuated at 628. For example, a motorized half-wave plate and apolarizing beam splitter with a shutter may be coupled to the pulsedlaser source 410 and/or 412, and used to attenuate the power of thepulsed laser beam to a range in the order of units of micro-Joules.

In some implementations, the cross-sectional area of the pulsed laserbeam is expanded at 630. For example, one or more optical elements, suchas 418 a or 418 b, may be used to adjust the cross-sectional area of thepulsed laser beam, which may be applied at the dispersive element 406.The optical elements 418 a and/or 418 b may expand the diameter of thepulsed laser beam from diameter in some implementations, while in otherimplementations, the optical element may contract the diameter of thepulsed laser beam, or adjust the shape of the pulsed laser beam in someother arbitrary form.

The pulsed laser beam is provided for manipulation, modification and/ordispersion at 632. For example, the second element 408 may image thepulsed laser beam dispersed by the dispersive element 406 in thebiological sample to form the multiphoton excitation area in thebiological sample. The shape and/or position of the multiphotonexcitation area in the biological sample may be adjusted.

The process 600C illustrated in FIG. 13 describes the detection andrecording of information about the dynamics of cellular or biochemicalprocesses of the biological sample based on the multiphoton excitationarea formed in the biological sample. In some implementations, theprocess 600C may be used in conjunction with the process 600A or theprocess 600B, or both, while in other implementations the differentprocesses may be used separately.

At 642, the signal associated with cellular dynamics of the biologicalsample is separated from the excitation light. For example, the secondelement 422 may separate a fluorescence signal corresponding to themolecular reporter used with the biological sample from the multiphotonexcitation area light generated by the pulsed laser beam.

The separated signal is propagated on to a detection plane at 644. Forexample, the third element 424 may propagate the signal separated by thesecond element 422 onto an imaging sensor of the detector 426.

At 646, information about the cellular or biochemical process dynamicsof the biological sample is captured at single-cell resolution. Forexample, the detector 426 may generate images of the dynamics ofcellular or biochemical processes of the biological sample using animage sensor. The images may be generated with a resolution in the orderof a single cell. These images may be captured using scripts andprocessed, or stored for post-processing.

The disclosed and other examples may be implemented as one or morecomputer program products, i.e., one or more modules of computer programinstructions encoded on a computer readable medium for execution by, orto control the operation of, data processing apparatus. Theimplementations can include single or distributed processing ofalgorithms. The computer readable medium can be a machine-readablestorage device, a machine-readable storage substrate, a memory device,or a combination of one or more them. The term “data processingapparatus” encompasses all apparatus, devices, and machines forprocessing data, including by way of example a programmable processor, acomputer, or multiple processors or computers. The apparatus caninclude, in addition to hardware, code executed by the that creates anexecution environment for the computer program in question, for example,but not limited to, code that constitutes processor firmware, a protocolstack, a database management system, an operating system, or acombination of one or more of them.

A system may encompass all apparatus, devices, and machines forprocessing data, including by way of example a programmable processor, acomputer, or multiple processors or computers. A system can include, inaddition to hardware, code executed by the hardware that creates anexecution environment for the computer program in question, for example,but not limited to, code that constitutes processor firmware, a protocolstack, a database management system, an operating system, or acombination of one or more of them.

A computer program (also known as a program, software, softwareapplication, script, or code) can be written in any form of programminglanguage, including compiled or interpreted languages, and it can bedeployed in any form, including as a standalone program or as a module,component, subroutine, or other unit suitable for use in a computingenvironment. A computer program does not necessarily correspond to afile in a file system. A program can be stored in a portion of a filethat holds other programs or data (for example, but not limited to, oneor more scripts stored in a markup language document), in a single filededicated to the program in question, or in multiple coordinated files(for example, but not limited to, files that store one or more modules,sub programs, or portions of code). A computer program can be deployedfor execution on one computer or on multiple computers that are locatedat one site or distributed across multiple sites and interconnected by acommunications network.

The processes and logic flows described in this document can beperformed by one or more programmable processors executing one or morecomputer programs to perform functions by operating on input data andgenerating output. The processes and logic flows can also be performedby, and apparatus can also be implemented as, special purpose logiccircuitry, for example, but not limited to, an FPGA (field programmablegate array) or an ASIC (application specific integrated circuit).

Processors suitable for the execution of a computer program include, byway of example, both general and special purpose microprocessors, andany one or more processors of any kind of digital computer. Generally, aprocessor will receive instructions and data from a read only memory, ora random access memory, or both. The essential elements of a computercan include a processor for performing instructions and one or morememory devices for storing instructions and data. Generally, a computercan also include, or be operatively coupled to receive data from ortransfer data to, or both, one or more mass storage devices for storingdata, for example, magnetic, magneto optical disks, or optical disks.However, a computer need not have such devices. Computer readable mediasuitable for storing computer program instructions and data can includeall forms of nonvolatile memory, media and memory devices, including byway of example semiconductor memory devices, for example, but notlimited to, EPROM, EEPROM, and flash memory devices; magnetic disks,such as internal hard disks or removable disks; magneto optical disks;and CD ROM and DVD-ROM disks. The processor and the memory can besupplemented by, or incorporated in, special purpose logic circuitry.

While this document may describe many specifics, these should not beconstrued as limitations on the scope of an invention that is claimed orof what may be claimed, but rather as descriptions of features specificto particular embodiments. Certain features that are described in thisdocument in the context of separate implementations can also beimplemented in combination in a single embodiment. Conversely, variousfeatures that are described in the context of a single implementationscan also be implemented in multiple implementations separately or in anysuitable sub-combination. Moreover, although features may be describedabove as acting in certain combinations and even initially claimed assuch, one or more features from a claimed combination in some cases canbe excised from the combination, and the claimed combination may bedirected to a sub-combination or a variation of a sub-combination.Similarly, while operations are depicted in the drawings in a particularorder, this should not be understood as requiring that such operationsbe performed in the particular order shown or in sequential order, orthat all illustrated operations be performed, to achieve desirableresults.

Only a few examples and implementations are disclosed. Variations,modifications, and enhancements to the described examples andimplementations and other implementations can be made based on what isdisclosed.

We claim:
 1. A device for recording the dynamics of cellular processes,the device comprising: one or more dispersive elements configured toreceive a pulsed laser beam with a spectrum of different wavelengths anddisperse the spectrum of the pulsed laser beam; one or more firstelements configured to: receive the dispersed spectrum of the pulsedlaser beam, and generate a multiphoton excitation area in a biologicalsample by re-overlapping in time and space the dispersed spectrum of thepulsed laser beam on an area in the biological sample; and a molecularreporter configured to artificially introduce labeling molecules intothe biological sample, wherein the device is configured to record athigh speed changes of cellular and biochemical processes of a populationof cells of the biological sample based on generation of the multiphotonexcitation area in the biological sample and subsequent optical readoutof light emission properties of the artificially-introduced labelingmolecules.
 2. The device of claim 1, wherein the high speed recordingincludes near-simultaneous imaging of the cellular or biochemicalchanges within a single plane of the biological sample.
 3. The device ofclaim 2, wherein the near-simultaneous imaging includes imaging in anorder of seconds to picoseconds.
 4. The device of claim 1, wherein themultiphoton excitation area includes a wide-field excitation disc withone of a predetermined diameter or a predetermined axial confinement,wherein the predetermined diameter or the predetermined axialconfinement is user-configurable, and wherein at least one of thepredetermined diameter or the predetermined axial confinement is in arange that is in an order of 1 μm to 1 mm.
 5. The device of claim 1,wherein a first element includes at least one of a grating, a prism, alens or a microscope objective lens that are configured to generate themultiphoton excitation area in the biological sample by imaging onto thebiological sample an area on the dispersive element illuminated by thepulsed laser beam.
 6. The device of claim 1, wherein the multiphotonexcitation area comprises a disc shaped excitation area of the dispersedspectrum of the pulsed laser beam, and wherein spectral components ofthe pulsed laser beam overlap in time and space in a focal region of thefirst element.
 7. The device of claim 1, further comprising: a firstamplifier that is configured to amplify the pulsed laser beam, whereinthe pulsed laser beam amplified by the first amplifier is used at thedispersive element; a wavelength tuning element that is configured totune the central wavelength of the pulsed laser beam amplified by thefirst amplifier to an excitation wavelength of the labeling moleculesartificially-introduced by the molecular reporter into the biologicalsample; and a motorized half-wave plate and a polarizing beam splitterwith a shutter that are configured to attenuate the power of the pulsedlaser beam at an output of the wavelength tuning element, wherein themolecular reporter is configured to facilitate recording of the cellularor biochemical changes of the biological sample.
 8. The device of claim7, wherein the first amplifier includes a regenerative amplifier.
 9. Amethod for high-speed imaging of cellular or biochemical changes of apopulation of cells of a biological sample, comprising: providing apulsed laser beam with a spectrum of different wavelengths at adispersive element; dispersing the spectrum of the pulsed laser beamusing the dispersive element; generating, using a first element, amultiphoton excitation area based on the dispersed spectrum of thepulsed laser beam; applying, by the first element, the multiphotonexcitation area to a biological sample; artificially introducinglabeling molecules into the biological sample; and capturing, using animaging detector array, information about the cellular or biochemicalchanges of a population of cells of the biological sample included inthe multiphoton excitation area by optically reading light emissionproperties of the artificially-introduced labeling molecules, whereinthe information about the cellular or biochemical changes are capturedin parallel at a resolution of a single cell.
 10. The method of claim 9,wherein providing the pulsed laser beam at the dispersive elementcomprises: generating the pulsed laser beam using a laser source;amplifying, using a first amplifier, the pulsed laser beam; tuning,using a wavelength tuning element, central wavelength of the pulsedlaser beam amplified by the first amplifier to an excitation wavelengthof the labeling molecules artificially-introduced by a molecularreporter that produces a signal associated with the cellular orbiochemical changes of the cells of the biological sample; and providingthe pulsed laser beam tuned by the wavelength tuning element at thedispersive element.
 11. The method of claim 10, wherein providing thepulsed laser beam tuned by the wavelength tuning element at thedispersive element further comprises: attenuating the power of thepulsed laser beam tuned by the wavelength tuning element using anattenuating element; modulating a cross-sectional area of the pulsedlaser beam using a second optical element; and providing the pulsedlaser beam with the modulated cross-sectional area at the dispersiveelement.
 12. The method of claim 11, wherein the attenuating elementincludes one of a half-wave plate and a beam splitter, or a shutter. 13.The method of claim 9, wherein the first element includes at least oneof a relay lens or a microscope objective that are configured togenerate the multiphoton excitation area in a focal region of themicroscope objective.
 14. The method of claim 9, wherein the labelingmolecules are configured to readout at least one of calciumconcentrations in the cells, or changes to calcium concentrations in thecells.
 15. The method of claim 14, wherein the labeling moleculescomprise a nuclear-localized, genetically encoded calcium indicatorwhich enables recording the dynamics of individual cells of thebiological sample.
 16. The method of claim 9, wherein the labelingmolecules comprise proteins that are expressed by the cells byintroducing genetic information to the cells.
 17. The method of claim 9,wherein the labeling molecules are configured to report on at least oneof membrane voltage of the cells, or changes to the membrane voltage ofthe cells.
 18. The method of claim 9, wherein the labeling molecules areconfigured to report on synaptic activity in the cells.
 19. The methodof claim 9, wherein the labeling molecules are configured to report onmetabolic activity in the cells.
 20. The method of claim 9, wherein thelabeling molecules are configured to report on enzymatic activity in thecells.
 21. The method of claim 9, wherein the labeling molecules areconfigured to bind to at least one of naturally available proteins inthe cells, or other molecules in the cells.
 22. The method of claim 9,wherein the labeling molecules are configured to report on a stage ofthe cell cycle.
 23. A device for recording the dynamics of cellularprocesses, the device comprising: one or more manipulating elementsconfigured to manipulate spatial or angular displacement of a pulsedlaser beam that includes a spectrum of different wavelengths; one ormore first elements configured to modify the shape of the pulsed laserbeam; one or more dispersive elements configured to disperse thespectrum of the pulsed laser beam; one or more second elementsconfigured to generate a multiphoton excitation area based on thedispersed spectrum of the pulsed laser beam and apply the multiphotonexcitation area to a biological sample; and a molecular reporterconfigured to artificially introduce labeling molecules into thebiological sample, wherein the device is configured to record athigh-speed changes of cellular or biochemical processes of a populationof cells of the biological sample based on application of themultiphoton excitation area to the biological sample and subsequentoptical readout of light emission properties of theartificially-introduced labeling molecules.
 24. The device of claim 23,wherein a first element includes a spherical lens that is configured toshape an area on the dispersive element used by the manipulating deviceto generate a trajectory on the dispersive element that corresponds tothe multiphoton excitation area in the biological sample.
 25. The deviceof claim 23, wherein the multiphoton excitation area is disc shaped witha predetermined axial confinement that is user-configurable in a rangein an order of 1 μm to 1 mm.
 26. The device of claim 25, wherein the oneor more second elements is configured to vary the diameter of the discshaped excitation area.
 27. The device of claim 23, wherein the deviceis configured to perform the high-speed recording at single-cell spatialresolution for a predetermined time period.
 28. A device comprising: oneor more manipulating elements configured to manipulate spatial orangular displacement of a pulsed laser beam that includes a spectrum ofdifferent wavelengths; one or more first elements configured to modifythe shape of the pulsed laser beam; one or more dispersive elementsconfigured to disperse the spectrum of the pulsed laser beam; and one ormore second elements configured to generate a multiphoton excitationarea based on the dispersed spectrum of the pulsed laser beam and applythe multiphoton excitation area to a biological sample, wherein thedevice is configured to record at high-speed changes of cellular orbiochemical processes of a population of cells of the biological samplebased on application of the multiphoton excitation area to thebiological sample, and wherein the device further comprises a detectionsection for detecting the changes of the cellular or biochemicalprocesses of the cells of the biological sample, the detection sectioncomprising: a third element that is configured to separate a signalassociated with the changes of the cellular or biochemical processesfrom the pulsed laser beam; and a fourth element that is configured topropagate the separated signal onto a detection plane of a detector. 29.The device of claim 28, wherein the detection section detector is animaging detector array that is configured to generate images of thecellular or biochemical changes of the population of cells of thebiological sample.